Compounds of albumen and cholesterol



Patented Jan. 1, 1946 COMPOUNDS OF ALBUMEN AND CHOLESTEROL Manfred Oberdiirfler, Berlin WI 50,

Adolf Biitenandt, Berlin-Dahlem, Joseph Schmidt- Thom, Berlin-Zehlendorf, and Hans Maier- Hiiser, Berlin N. 65, Germany, asslgnors to Schering Corporation, Bloomfield, N. 3., a corporatlon of New Jersey No Drawing. Application Aprll'4, 1941, Serial No. 386,922. In Germany and Switzerland April 8,

2 Claims.

This invention relates to compounds of albumen and cholesterol and a process for makin said compounds and their solutions.

It is already known that aqueous extracts or extracts with bufier solutions of organs or body liquids or preparations, as for instance, an organ pulp can be precipitated by means of neutral salts, in order to isolate therapeutically valuable substances from the precipitate or filtrate. Thus, for instance, hormones, ferments, antigenes and anti-bodies have been prepared in a very pure state.

7 It was, however, not known that a new group of compounds, namely compounds of albumen and cholesterol may be obtained by using the above mentioned methods.

Thus, therapeutically valuable albumen-cholesterol compounds or complexes or their solutions can be obtained, for instance, by subjecting fresh, frozen or dried organs or body liquids, which contain the cholesterol-albumen complex, to a careful and mild extraction with solvents, not able to denature albumen, for instance, with water or buffer solutions. Thereby it is advisable to keep the pH of the solutions at about the neutral point, that is to say, at about 7. It is also possible, if necessary, to work under slightly acid or alkaline conditions, by selecting suitable solvents. The optimum of the pH value of the solutions, however, lies between about 6.0 and 7.6. It is of advantage to use fresh organs, if possible.

As starting materials there may be used especially suprarenal glands, liver and also blood serum. Such organs can be very readily obtained, for instance, from Warm-blooded animals. such as cattle, horses, sheep, pigs, rabbits and so on. Besides the above mentioned organs other organs may also be employed, although not as successfully, likewise human body liquids may be used for the above mentioned purpose. The organs are preferably cut into very small pieces. The pulp obtained is then extracted thoroughly in the cold with water or with the buifer solution, for instance, by shaking the same in a suitable apparatus for several hours. Under certain conditions it may be advantageous to carry out the working up at room temperature or even at body temperature.

As bufier solutions, for instance, {6 molar phosphate, citrate or Veronal (sodium salt of diethyl barbituric acid)buffers according to Michaelis are suitable. ,These three mentioned buffer solutions have been found to give similar results at a pH of 7.0. Changing the buffer solution instead of using the same solution, in case the treatment of the precipitates with the bufler mixtures is repeated, is without any importance. A change of the pH of the buffer solutions, as for instance, the successive use of phosphate buffer solutions of pH 6.4, 7.0, and 7.6 within one series of precipitations, however, proves to have a favorable effect; thereby the order of applying said solutione with the difierent pHs is not important.

The solutions, which are thus employed for the extraction of the organs, are preferably mixed with a suitable disinfectant. for instance, with phenyl mercuric nitrate in a proportion of 1: 1,000,000.

It is sometimes of advantage to add to the extraction liquids and also to the buffer solutions suitable water soluble organic solvents, as for instance, alcohol, glycerol, glycol, dioxane and the like. Such additions are suitably used in an amount of not more than 10% of the buffer solutlon.

After removing the solid particles, for instance, by pouring the extracts obtained through a coarse sieve, albuminous fractions are obtained from said extracts, according to methods known per se, and from said fractions those fractions are isolated, which possess thehighest saponine detoxicating eifect. For this purpose the extracts or solutions are treated, for instance, with a neutral albumen precipitating liquid, which is capable of reversibly precipitating the albumen-cholesterol-complcx, that is to say, which does not denature the albumen; an especially suitable method consists in salting out said extracts with suitable salts, such as ammonium sulphate. The precipitate, thus obtained, is separated from the liquid, for instance, by centrifuging after having been allowed to stand for several hours. The precipitate may be further purified by dissolving the same, for instance, in a buifer solution whereby the active principle is dissolved while the impurities remain undissolved, and then precipitating it again, for instance, with ammonium sulphate. If necessary, this process is repeated several times. Toxic water soluble components, which sometimes may be present in the precipitate-this depends on the organs, which were employed as starting materialas for instance, adrenalin in the 'suprarenal gland, histamine in the lungs, etc., can be eliminated by methods known per se, preferably by suitable adsorption agents, whereby, however,

those with strongly acid or alkaline reaction are not to be employed. The precipitation can also be effected by convertlngthe compound to be precipitated into suitable difllcultly soluble or insoluble salts, for instance, into alkaline earth or heavy metal salts. which can again be decomposed in known manner, so that the metal-free albumen-cholesterol-compler. is regenerated.

Finally, the impurities or noxious substances resent in the extracts of or ans or the body liquids can be fractionally precipitated. so that the desired albiimen-cholesterol-comnound remains dissolved. If necessary. these modes of procedure mav be combined with each other.

The degree of purity of the complex compound obta n d can be determinedby means of a test using the detoxicating efl'ect upon saponines, such as digitonine. such as the test whereby the hemolytic effect of saponines is counteracted by the cholesterol-albumen-compound.

A verv suitable methodoi' determining the saponine-detoxicatlng effect is based upon the fact that red blood corpuscles how hemolysls on ning into a saponine solution, for instance. into a digitonine solution. First one observes. on allowing blood or blood corpuscles into an isotonic digitonine solution of about 0.01%, that the liquid becomes turbid by erythrocytes. After a short period of time the solut on becomes clear (of the color of lacquer) on account of hemolvsis taking place rapidly. Most probably the digitonine is thereby combined with the cholesterol of the blood corpuscles. On further addition of blood corpuscles drop by drop the becoming turbid and clear of the solution repeats itself until all the digitonine is combined. Thereafter the solution remainspermanently turbid upon adding further red blood corpuscles.

In order to make use quantitatively of this effect it is advisable to proceed as follows: The blood or the red blood corpuscles are allowed to flow from a micro-burette into the digitonine solution to be tested. Preferably there are used for titration washed blood corpuscles obtained by centrifuging citrate blood (containing 60 mgs. of solid sodium citrate in cos. of venous blood). removing the supernatant plasma. washing the blood corpuscles several times with an isotonic sodium chloride solution. and centrifuging again. Finally the centrifuged citrated blood corpuscles are suspended in isotonic sodium chloride solution. so as to yield the same amountof ccs. as before removine the plasma. For the titra ion the washed blood corpuscles obtained in this manner are preferably diluted w th isotonic solution in the proportion of 1:10. Upon allowing such a suspension or red blood corpuscles to flow into 1 cc. of an about 0.01% digitonine solution, about 0.5 to 0.8 cc. of the same are required in order to yield a lasting turbidity. The titration is prefer-. ably carried out with freshly prepared washed blood corpuscles and at a temperature of 40 C., in order to eliminate errors.

Cholesterol and digitonine form a diificulty soluble molecular compound consisting of 1 mol of cholesterol and 1 mol of digitonlne. Hence, it follows that 1 part by weight of cholesterol is able to combine with 3.18 parts by weight of digitonine or 1 part by weight of digitonine with 0.314 part by weight of cholesterol.

For determining the saponine detoxicating effect of any solution, such as a solution of an albumen-cholesterol-complex, by means of this method one proceeds as follows: First the hemolytic index of the suspension of blood corpuscles, i. e. that amount of the suspension which is caused to be hemolyzed by a certain amount of digitonine, is determined. Then the solution to be tested for its hemolysis preventing eil'ect is added to a known amount of the digitonine solution. By the presence of a saponine detoxicating compound in the solution to be tested part of the digitonine is fixed. Therefore the subsequent titration with the same suspension of blood corpuscles requires a smaller amount of the latter in order to cause turbidity The diiference between the hemolytic index of the blood corpuscles before and after addition of the saponine detoxicating compound enables one to calculate the same in amounts of cholesterol contained in the solution to be tested.

Thus, by this method it is very easy to determine the saponine detoxicatlng eifect of any solution to be tested. Of course, other methods may be likewise used for this purpose.

The cholesterolalbumen-compounds obtained are very sensitive substances, thus, the albumenis split oil? after dialysing the compound for 12 hours against flowing or standing water. And the water-clear solution still containing considerable amounts of albumen does not show any effect in the hemolysis-preventing test, while the albumen recipitated, which is soluble neither in water nor in m./100 or m./15 phosphate bufler solution of pH 7, gives a positive hemolysis-preventing reaction in suspension.

Likewise, the compounds obtained according to this process do not stand an heat treatment or treatment with organic solvents capable of denaturing albumen, as for instance, alcohol of high percentage. From their aqueous solutions the compounds are precipitated by addition of salts, especially of ammonium sulphate.

It may be repeated that the gist of this invention consists in dividing an aqueous extract from organs or a body liquid containing albumen and cholesterol, into several fractions by methods known per se such as precipitation, adsorption, conversion into diiflcultly soluble compounds and the like, separating the fraction or fractions showing the highest detoxicating effect on saponines from the other fractions or the remaining liquid, and if necessary, repeating said fractionating processes one or several times, whereafter the remaining albumen-cholesterol-complex compound is isolated either in solid water-soluble form or as solution. Applicants are aware that it is already known that extracts of organs with water or buffer solutions have been subjected to fractionating. But in contrast to the present invention one has never selected and worked upthose fractions which show the highest saponinedetoxicating effect.

The following examples serve to illustrate the invention without, however, limiting the same to them:

Example 1 1 kg. of raw, preferably not previously frozen, fresh suprarenal glands or livers is passed through a meat mincing machine after being cut to small pieces. The minced meat is then placed into a container adapted to be shaken, together with 3 liters of 1% N phosphate buffer solution of pH 7 whereupon it is shaken therein for 8 hours. The entire mass is poured through a coarse sieve and then /3 of the volume in grams of pure ammonium sulphate is added to the nitrate. After allowing the mixture to stand for '3 hours, it is centrifuged for 40 minutes, the

mother liquor is decanted, and the residue dissolved in 1 liter of a boiler solution of DH=7 while stirring. Thereafter the solution is centrii'uged again for 40 minutes, the liquid obtained is decanted and mixed with 200 grs. of ammonium sulphate. This treatment is repeated twice. After the last centrifuging the mother liquor is decanted and the precipitate obtained by salting out with ammonium sulfate is dissolved in 100 cos. of a bufler solution of pH 7. The solution 'is then subjected to dialysis against 10 liters of a buifer solution of pH '1 for 12 hours. To 1 liter of the standard buffer solution there has been added previously 1 mg. of phenyl mercuric nitrate as d sinfectant.

The solution of the last precipitate obtained is tested with respect to its hemolysis preventing property as described above or as follows:

Into each of 10 small tubes 1 cc. of an 0.01% solution of digitonine in physiological sodium chloride solution is filled by means of a pipette, Thereto from the solution to be tested diminishing amounts of 1, 0.8, 0.6, 0.4, 0.2. 0.1, 0.05 cc. are added by means of a pipette. The tubes are then shaken and filled up with physiological sodium chloride solution, so that they all amount to 2 cos. After allowing the tubes to stand for 2 hours, each tube is mixed with 1 cc. of a 5% suspension of defibrinated mutton blood in physiological sodium chloride solution. The minimum amount of the solution to be tested which inhibits all visible hemolysis by the fixed amount of digitonine is regarded as the "hemolysis-preventing value" of the solution.

Example 2 100 grs. of beef liver are passed several times through a meat mincing machine; the thin pulp obtained is then extracted with 300 cos. of a A molar phosphate buffer solution of pH 37.0 while shaking. The tissue residue is removed by pressing out through cloth; the solution is then mixed with 100 grs. of finely pulverized solid ammonium sulfate in order to precipitat the albuminous fractions, while shaking vigorously. After allowing the mixture to stand for 30 minutes it is centrifuged, the liquid poured away, the precipitated albuminous fraction again dissolved in 300 cos. of the same buffer solution and again centrifuged, in order to separate those constituents oi the precipitate which are not dissolved. Thereafter the solution is tested for its cholesterol content by means of the above de- In all experiments carried out hitherto the test shows a content of about '15 v of cholesterol for the co. in the solutions, obtained according to these directions.

Example 3 100 grs. of a thin pulp of beef suprar'enal glands ar extracted with 300 cos. of a A molar phosphate buffer solution of pH 7.0 while adding 30 cos. of glycerol and shaking for 4 hours. After pressing the mass out through cloth the solution obtained is mixed with 100 grs. of finely pulverized ammonium sulfate inorder to precipitate the albuminous fraction. The precipitate, obtained on centrifuging, is again dissolved in 300 cos. of buffer solution. The constituents, which are not dissolved. are removed by contrifuging. The precipitation and dissolving are then repeated as described in the preceding examples.

The finely obtained solution shows in the he-' molysis preventing test a cholesterol cantent of 75 'y for the co.

Example 4 100 grs. of a thin pulp of bull testicles are extracted by shaking with 300 cos. of a molar phosphate buffer solution of pH 7.0 for 4 hours. After pressing the mass through cloth, the solution is mixed with 100 grs. of finely pulverized ammonium sulfate in order to precipitate the albuminous fraction. The precipitate, obtained on centrifuging, is again absorbed with 300 cos. of the same bufier solution. The constituents, which are not dissolved, are removed by centrifuging. This treatment is repeated as described in the preceding examples in order to further purify the product. The solution thus obtained shows in the hemolysis preventing test a choles terol content of 100 'y for the cc.

Example 5 Example 6 270 cos. of defibrinated pig'blood serum, which has been freed from blood corpuscles by centri-.

fuging the blood, are filled up to 1000 cos. with a molar buffer solution of pH '7 and then mixed with 300 grs. of ammonium sulfate. The albuminous precipitate is centrifuged, the supernatant liquid is decanted, and theresidue again dissolved in 250 cos. of the above mentioned buffer solution. The solution obtained is precipitated by means of '75 grs. of ammonium sulfate, the albuminous precipitate removed by centrifuging and again dissolved as described, wherea-fter the precipitation is repeated. The cholesterol-albumen-solution obtained prevents hemolysis with 4 cos, corresponding to a content of 7.5 mgs. of

cholesterol in cos. of the solution.

Example 7 pH=7, the solution subjected to a dialysis against liters of the same phosphate buffer solution for 16 hours and centrifuged. The dialyzed solution obtained, amounting to 1.26 liters of pH=6.6, is again precipitated by the addition of 1100 ccs. of 33% ammonium sulfate solution while stirring, the mixture is allowed to stand over night at room temperature, centrifuged, the residue dissolved in 300 cos. of the same buffer solution, and again subjected to dialysis against 10 liters of buffer solution over night. The dialyzed liquid is then centrifuged, 200 cos. of the liquid obtained are slowly mixed with 31.2 ccs. of acetone while stirring. After allowing the mixture to stand for 30 minutes, the precipitate is collected by centrifuging. It is freed from acetone by placing it into a high-vacuum over silicagel for one hour, dissolved in a buffer solution of pH 7 and centrifuged. The'solution obtained shows a strong hemolysis preventing effect.

On using, instead of acetone, the same amount of alcohol and proceeding in the same manner, a similar product is obtained.

The compounds obtained according to these examples are complex compounds containing cholesterol and albumen in the proportion of about 1:20-40. They are soluble, in water in the presence of neutral electrolytes and are readily split up into their components by strong acids and alkalis as well as organic solvents capable of denaturing proteins.

Of course, many changes and variations in the reaction conditions, the solvents and precipitating agents and the like, may be made by those skilled in the art in accordance with the principles set forth herein and in the claims annexed hereto.

. It may be repeated that all those methods of precipitation and purification have to be avoided whereby cholesterol and/or albumen is split off from the complex. Thus, for instance, dialysis or electrophoresis must not -be carried out too long, since then the complex is split. It is, however, comparatively easy to find out the optimum conditions by preliminary experiments wherein the results achieved by the various methods and conditions of reaction are followed up by testing the products for their saponine detoxicating effect, such as their hemolysis preventing power.

For salting out not only ammonium sulfate may be used but also other readily water soluble neutral salts'such as magnesium sulfate, sodium chloride and the like, although ammonium sulfate has proved to give the best results. The optimum amount of salts to be added to the solution for salting out the cholesterol albumencomplex, may also be found out by preliminary experiments thereby making use of the test methods mentioned. In the case of ammonium sulfate a concentration of -30%, i. e. about half saturation, has proved to yield the purest products. The salt present in the precipitate or its solution may be removed not only by dialysis, but also by other methods, such as electrophoresis, ultrafiltration and the like, whereby care has to be taken that the electrolytes are not completely removed from the solution, since then decomposition of the complex may take place.

One of the most important points in preparing the new cholesterol-albumen-compiex is the maintenance of the proper pH-value which, as stated above, must be around the neutral point.

I Repeated reprecipitatlon seems to be of great effect, since thereby the accompanying ballast compounds of albuminous character and with almost the same physical properties are removed. Thereby it is even possible, especially when using pig ovaries as starting material, to obtain completely clear solutions of the cholesterol-albumencomplex. Many other precautions have to be taken in order to obtain optimum results, as they are known to an expert in the field of protein chemistry, especially in the chemistry of globulines and other albuminous matter. Especially the methods used for isolating and purifying globulines have proved of advantage for the isolation and purification of the cholesterol aibumen-complex compounds.-

What we claim is:

'1. Concentrates of albumen-cholesterol complexes which are soluble in water in the presence of neutral electrolytes and are readily split up into their components by strong acids and alkalies as well as organic solvents capable of denaturing proteins.

2. Albumen-cholesterol complexes containing cholesterol and albumen in the proportion by weight of about 1:20-40.

MANFRED OBERDORFFER. anom- B'UTENANDT. JOSEPH scnMmr-momt. HANS MAIER-H'USER. 

